Cowpea Mosaic Virus Nanoparticles for Cancer Imaging and Therapy

Home , Comovirus, Cowpea mosaic virus, Secoviridae

Advanced Drug Delivery Reviews 145 (2019) 130–144

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Advanced Drug Delivery Reviews

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Cowpea mosaic virus nanoparticles for cancer imaging and therapy

Perrin H. Beatty, John D. Lewis ⁎

Department of Oncology, University of Alberta, Edmonton, Alberta T6G 2E1, Canada article info abstract

Article history: Nanoparticle platforms are particularly attractive for theranostic applications due to their capacity for Received 16 June 2018 multifunctionality and multivalency. Some of the most promising nano-scale scaffold systems have been co- Received in revised form 7 December 2018 opted from nature including plant viruses such as cowpea mosaic virus (CPMV). The use of plant viruses like Accepted 15 April 2019 CPMV as viral nanoparticles is advantageous for many reasons; they are non-infectious and nontoxic to humans Available online 17 April 2019 and safe for use in intravital imaging and drug delivery. The CPMV capsid icosahedral shape allows for enhanced ﬁ Keywords: multifunctional group display and the ability to carry speci c cargoes. The native tropism of CPMV for cell- Cancer therapy surface displayed vimentin and the enhanced permeability and retention effect allow them to preferentially ex- Cowpea mosaic virus travasate from tumor neovasculature and efﬁciently penetrate tumors. Furthermore, CPMVs can be engineered CPMV via several straightforward chemistries to display targeting and imaging moieties on external, addressable res- Drug delivery idues and they can be loaded internally with therapeutic drug cargoes. These qualities make them highly effec- eCPMV tive as biocompatible platforms for tumor targeting, intravital imaging and cancer therapy. Intravital imaging © 2019 Published by Elsevier B.V. Molecular targeting Non-invasive imaging Multifunctional Virus-like particle

Contents

1. Introduction...... 131 2. Characteristicsofthecowpeamosaicvirus...... 131 2.1. CPMVbiocontainment,biodistributionandpathology...... 132 2.2. Chemicalbioconjugation...... 134 2.3. ShieldingofCPMVnanoparticlesforincreasedbioretention...... 136 2.4. Geneticengineeringtointroducefunctionality...... 136 2.5. ProductionofCPMVandCPMVvirus-likeparticles...... 136 3. TherapeuticandtheranosticusesofCPMVs...... 137 3.1. Tumorhomingpeptides...... 137 3.2. Cargoloading...... 137

3.3. Photodynamic therapy using CPMV-C60 fullereneconjugates...... 138 3.4. UseofCPMVasavaccineincancerimmunotherapy...... 138 4. Intravitalvascularimagingfornon-invasivecancerdetection...... 139 4.1. Chickembryonicchorioallantoicmembranemodels...... 139 4.2. Deep tissue imaging using CPMVs decorated with multivalent ﬂuorescentdyes...... 139 4.3. NativetropismofCPMVforvimentinonhostcells...... 140 4.4. CPMVtargetedtogastrin-releasingpeptidereceptors...... 141 4.5. Neovascularimagingviavascularendothelialgrowthfactorreceptor(VEGFR)targeting...... 141 4.6. Neovascularimagingviaepidermalgrowthfactor-likedomain7(EGFL7)proteintargeting...... 141 5. Conclusionsandfuturedirections...... 142 Acknowledgements...... 142 References...... 142

⁎ Corresponding author at: Translational Prostate Cancer Research Group, Department of Oncology, University of Alberta, 5-142C Katz Group Building, 114th St and 87th Ave, Edmonton AB T6G 2E1, Canada. E-mail address: [email protected] (J.D. Lewis).

1. Introduction Table 1 Advantageous features of CPMV for use in tumor cell imaging and therapy.

Many different scaffold and carrier systems have been co-opted from Features Measurement or description Reference nature, or synthetically designed for use in tumor cell-specific imaging Non-pathogenic and Dosages up to 100 mg/kg body weight in [23,27] – and drug delivery (reviewed in [1 7]). Naturally-occurring scaffold- non-toxic mice (1016 CPMVs) are nontoxic. carriers include viral nanoparticles (VNP) and self-assembling protein Biodistribution After dosing in mice, found in various organs, [40,47] cages [7–13]. VNPs such as temperate filamentous bacteriophage (M13, culminating in the liver and spleen. fd), lytic capsid and tailed bacteriophage (T4, P22, λ etc.) or plant viruses; Biocompatible and CPMV were retained for 72 h in chick embryo [23] long retention endothelium system and in mice for 1 to brome mosaic virus (BMV), red clover necrotic mosaic virus (RCNMV), several days. potato virus X (PVX), tobacco mosaic virus (TMV), cowpea chlorotic Biodegradable CPMVs do not persist in vivo, therefore they [55] mottle virus (CCMV), cowpea mosaic virus (CPMV) and mammalian vi- are good candidates for therapeutic use. ruses; canine parvovirus (CPV), influenza A and hepatitis B have been Physical and chemical Temperature: up to 60C, pH range 3 to 9, [7,33,40] stability organic solvents such as dimethyl sulfoxide, used as scaffolds and cargo-carriers in cancer research [7,9–11,14–18]. resistant to proteolysis and gastric and Supramolecular, self-assembled protein cages such as heat shock protein intestinal conditions. (Hsp), ferritin, and vault have also been utilized as drug delivery nano- High resolution and Dye-labelled CPMV nanoparticles did not [23] particles [8,19]. In addition, medically relevant, organic and inorganic non-aggregating aggregate, allowing for high resolution synthetic nanoparticles have been designed; nanobombs, nanoworms, imaging. Fully characterized RNA-1 and -2 genomes and gene products [32,57,118] micelles, liposomes, dendrimers, dendrons, superparamagnetic iron sequenced and annotated. X-ray oxide nanoparticles, gold nanoparticles and quantum dots [5,20–22]. Al- crystallography and cryogenic electron though these various carriers differ in shape and macromolecular com- microscopy done on capsid ponents they share three essential features; (1) consist of uniform size In vivo production of CPMVs with encapsidated RNA genomes. [7] CPMV High yield: 0.8 to 1.0 mg CPMV per g infected distribution within their type, (2) act as molecular scaffolds to display cowpea leave tissue different functional moieties, and (3) act as drug delivery vehicles due In vitro production of CPMV VLPs via trans expression of L-S [28,82,89] to their internal cargo carrying capacity [5,7]. Possibly the largest advan- eCPMV subunit fusion protein and 24 K protease in tage of using any of these nanoparticles in cancer research, diagnosis and cowpea protoplasts or insect cells. High therapy is their multifunctionality. Nanoparticles, including VNPs like yield: 1 g pure eCPMV per kg fresh-weight N. benthamiana leaf tissue CPMV, can simultaneously display imaging probes, targeting or homing Ease of Synthesis and purification of dye-labelled [57] moieties and carry a chemotherapy drug as cargo for maximum anti- functionalization CPMV nanoparticles in one day, using either cancer activity [5]. For example, non-invasive, intravital, vascular imag- standard or click chemistry ing has been problematic as a tumor detection and diagnostic tool External conjugation 60 asymmetrical protein units per capsid [33,57] because of inadequate resolution and poor fluorescent dye tissue pene- with, lysine with 5 solvent-exposed lysine residues per residues protein unit, providing a total of 300 tration [23]. However, the VNP scaffold multifunctionality allows for potential conjugation sites per nanoparticle. high density display of fluorescent dye molecules and targeting ligands Single functional CPMV labelled with multiple copies of a [57] simultaneously with the ease of VNP extravasation through leaky group display single functional group (e.g. 120 copies of fl tumor blood vessels [24]. The fluorescent nanoparticles remain bright, uorescent dye) made with no detriment to the signal. without detectable quenching, which increases the resolution, and can Multiple functional CPMV labelled with single copies of multiple [57] target deep tissue vascular endothelial cells for up to 72 h [23,25]. group display functional groups made with no detriment to Which carrier system to use for tumor imaging and or drug delivery targeting. depends on the physiochemical and pharmacokinetic properties of the Native tropism WT-CPMV nanoparticles bind to vimentin on [112] carrier, biological distribution of the tumor, immunogenicity between endothelial cells Internal conjugation The thiol side chains of 2 cysteine residues [112] the carrier and host, and the ratio of toxicity between host diseased are ligation handles for internal conjugation. cells to host healthy cells. Cargo loaded capsid 130 to 155 dye or drug molecules per CPMV [27] There are many features of CPMV nanoparticles that make them capacity via nanoparticle. good candidates for both chemical and genetic engineering for develop- infusion Cargo loaded capsid Cargo-loaded CPMVs stored in buffered [27] ment as cancer imaging and therapy tools. These features include their storage stability saline, pH 7 at 4 °C were stably encapsulated non-pathogenicity, biocompatibility, non-aggregation and biodegrad- for weeks. ability in mammalian systems, temperature and pH stability, ease of ex- Deep tissue Intravital imaging of large vessels to a depth [23,57] ternal functionalization by either single or multiple functional group visualization of 500 μm or microvasculature to 200–250 μ display, cargo loading capacity, native tropism towards vimentin on en- m for up to 72 h. In vitro studies Yolk sac and embryonic vasculature [107] dothelial cells and their native immunostimulatory effect within solid (vascular endothelium and blood flow) of tumor cancer models. In addition, CPMVs have been fully characterized explanted mouse embryos. Various plate by X-Ray crystallography, electron microscopy and genome sequencing assays. and protocols have been developed to produce CPMVs using in vitro and In vivo studies Tumor cell xenografts of chick embryo [23] chorioallantoic membrane and adult mouse in vivo methods. This review discusses some of the uses of CPMV vascular endothelium. nanoparticles for the use in research and development of diagnostic, Immuno-stimulatory CPMV can induce potent antitumor immune [107] therapeutic and theranostic cancer tools. Table 1 lists some of the ad- effect response after in situ vaccination in models vantageous features of CPMV for use in tumor cell imaging and therapy. of skin, ovarian, breast, and colon cancers Table 2 highlights some examples of functional group classes that have CPMV may also eliminate M2 macrophages. been reported in the literature as being conjugated to CPMV. approximately 30 nm in diameter, with a net negative surface charge 2. Characteristics of the cowpea mosaic virus and a molecular weight of 5.6 × 106 g mol−1 (Fig. 1)[3,11,19,23,26]. The capsid shape provides a large surface area to volume ratio, which The cowpea mosaic virus (CPMV) is a picorna-like virus of the order is advantageous for enhanced multifunctional group display and cargo Picornavirales,familySecoviridae and genus Comoviridae that naturally (or payload) carrying capacity [27]. CPMV has a bipartite, positive- infects the black-eyed pea plant Vigna unguiculata.CPMVisanisomet- sense RNA genome comprised of RNA-1 (5.89 kb) and RNA-2 ric, icosahedral lattice capsid with pT = 3 quasi symmetry, (3.48 kb) molecules that are encapsidated separately [19,27–29]. As 132 P.H. Beatty, J.D. Lewis / Advanced Drug Delivery Reviews 145 (2019) 130–144

Table 2 such as V. unguiculata act as vectors to transmit CPMV from host plant Some examples of functional group classes conjugated to CPMV capsids. to host plant, with the severity of plant host infection positively corre- Class of functional group Description Reference lating with the beetle feeding rate [34]. There is no evidence that the carrier beetles contract disease from CPMV. Viable CPMV infection of Shielding groups V. unguiculata is only mediated when both B and M nanoparticles are in- Polyethylene glycol Inert polymers that add an [7,19,23,33,73] troduced into plant leaves or stems together, and the empty particles immunity-response shielding are non-infectious [27]. ‘stealth layer’ to the particles. Carbohydrates, lipids Alternative, nature-inspired [75] and proteins shielding materials. 2.1. CPMV biocontainment, biodistribution and pathology

Imaging groups The approval of the use of viral-based therapeutics in humans in

Fluorescent dye Alexa Fluor 647, A555, Oregon [20,23,40] North America has been slow due to concern that the viruses, even at- molecules Green 488 tenuated versions, could develop higher virulence, adapt to new hosts PEG500f Peptide with a fluorescein, [54] or revert to wildtype because of their high mutation and recombination monodisperse PEG polymer and rates [35]. Mammalian viruses like adenovirus, herpesvirus and lentivi- hydrazido terminus. rus have been developed as successful therapeutic agents against multi- Gadolinium ions Contrasting agent platforms. [55,64] CPMVs can be decorated with ple different diseases in humans (reviewed in [36]). However there hundreds of Gd3+ ions. have been examples of these viral-based therapies causing harm or even death in treated patients [37]. Concerns have also been raised Targeting/homing groups about using RNA-virus based therapeutics due to the potential for acti- Vimentin CPMV shows natural targeting of [54,99,109,110,112] vation of human proto-oncogenes or inactivation of tumor-suppressor surface exposed membrane genes via mutagenesis from viral RNA integration into the human vimentin. genome [6]. Bacteriophage and plant virus-based VNPs, unlike Folic acid Folic acid ligand targets cancer [100,101] cells in vitro. mammalian viruses, are non-infectious towards mammals and so are Human-holo-transferrin Targets transferrin receptors that [67] considered nontoxic to humans and safe for use in intravital tumor protein are overexpressed on a variety of cell imaging and drug delivery. However, CPMV is a picorna-like virus cancer cells. similar in genetic synteny and protein capsid structure with animal pi- Pan-bombesin peptides Targets gastrin-releasing peptide [20] cornaviruses such as polio viruses, coxsackie viruses and Theiler's mu- receptors overexpressed on prostate carcinoma cells. rine encephalomyelitis virus [38]. Certainly, there are some viruses Peptide F56 Targets specifically to VEGFR-1 [54] that have host ranges that include plants and animals [39]. As well, receptors on tumor endothelial CPMV particles will bind to the surface of vertebrate endothelial cells cells. and become internalized into the cells, although they cannot replicate. E7p72 peptides Targets EGFL7 protein expressed [25] by endothelial cells undergoing This feature allows the virus to be used as an imaging and drug delivery vascular remodelling and not by nanoparticle [20]. CPMV particles also remain intact after administra- quiescent cells. tion in vivo, even through the gastrointestinal tract, therefore there is complex sugars, Variety of other molecules [2,11,58,67,69] a potential path for CPMV leakage from experimental animals or pa- peptides conjugated to CPMV. Reviewed in tients to plants and the environment [28,40–43]. Although CPMV parti- references listed. cles recovered from mice tissues or incubated with plasma or serum Therapeutic groups were unable to cause an infection cycle in plants [43]. Taken together, although the risks are very low, it is considered prudent to protect pa- C60 Buckyball Photosensitizer. [103] Zinc ethynylphenyl Photosensitizer, conjugated to [84] tients, humans and the environment from inadvertent mutation, new porphyrin CPMV-dendron hybrid host adaptation, off-target effects and escape into the environment nanoparticle. from therapeutic use of CPMV therefore bio-containment measures should be employed with in vivo CPMV therapy [6,44]. well, approximately 10% of wildtype (WT), intact, viral particles can be CPMV is resistant to most of the common viral attenuation protocols, found empty of RNA [19]. Density gradient centrifugation separates however, exposure to 254 nm UV radiation for short time doses was suf- these CPMV particles into a top (T), middle (M) and bottom band ficient to crosslink the encapsidated RNA genome, and therefore inacti- (B) that are; empty particles (CPMV-T), RNA-2 carrying particles vate the virus, while still retaining protein structure and function (CPMV-M) and RNA-1 carrying particles (CPMV-B), respectively [19,42,45]. However, since in planta CPMV infection necessitates the [27,30]. presence of the bipartite RNA genomes within the capsids to allow for RNA-1 encodes: proteinase K cofactor (ProC), helicase, virus expression of the viral gene products needed for replication and capsid genome-linked protein (Vpg), 24K proteinase and RNA-dependant production, removing the RNA genomes would render CPMV non- RNA polymerase. RNA-2 encodes, 48/58 K movement protein and replicative [46]. The use of non-replicative, CPMVs would obviate the VP60 which is post-translationally cleaved by the RNA-1 encoded 24K need for bio-containment and therefore allow therapeutic usage of protease to form the two capsid protein subunits; L and S (Fig. 2). medically effective CPMVs in humans. Of course, these non-replicative Each linear RNA genome has a Vpg cap at the 5′ end and a polyaden- CPMV versions need to maintain the capsid structure, function and ylation tail at the 3′ end [31]. The two capsid protein subunits are called chemical reactivity of the WT-CPMV nanoparticles [42,45]. the small (S, 24 kD) and large (L, 42 kD) subunits, with one (A) and two The plasma clearing, biodistribution and toxicity of CPMV in mice jelly roll β-barrel domains (B and C), respectively [11,19,32,33]. This dosed either orally or intravenously and in chick embryos intravenously jelly roll β-barrel polypeptide structure of two twisted, antiparallel β- has been studied with varying results [23,40,47]. CPMV inoculated into sheets, is common amongst Comoviruses and presents as externally chick embryos was detected and internalized within the vascular endo- and internally extended loops on the capsid [19,26]. Together, domains theliumsystem[23]. In mice intravenously dosed with 100 μgoffluores- A, B and C, comprise one asymmetrical unit with each capsid containing cently labelled CPMV, the nanoparticles were detected in the spleen, 60 units total (Fig. 1)[11]. kidney, liver, lung, stomach, small intestine, lymph nodes, bone marrow, In nature, beetles from the Subfamily Galerucinae and Family vascular endothelium and brain for several days after administration Chrysomelidae that feed on the leaves and stems of virus-host legumes [40]. Oral dosage of the same amount and labelling of CPMV in mice P.H. Beatty, J.D. Lewis / Advanced Drug Delivery Reviews 145 (2019) 130–144 133

Fig. 1. CPMV images. (a) Subunit organization of capsid at 3 Angstrom resolution, (b) CPMV T = pT3 lattice scaffold, (c) Cut-away inside view of CPMV, (d) the one domain of the S (small) subunit (blue) and the two domains of the L (large) subunit (red). The S and L subunits comprise one asymmetrical unit. The CPMV capsid is made of 60 asymmetrical units. The inset image shows the location of one asymmetrical unit on the capsid. The ﬁve addressable lysine residues that are commonly used as ligation handles are labelled. The ﬁgures/data were obtained from VIPERdb (http://viperdb.scripps.edu). [119].

Fig. 2. (a) The CPMV bipartite RNA genome organization. The arrow in RNA-2 points to the location in the nucleic acid sequence that encodes the βB-βC loop in the S subunit, which is a site for insertion of a nucleic acid sequence encoding a peptide to be externally displayed on the capsid. (b) Two vectors that were designed to allow high expression levels of the VP60 and the 24K protease genes in planta, to produce eCPMV, together these vectors are termed CPMV-HT. (c) pEAQexpress-VP60-24K is a vector designed for transformation into N. benthamiana plants for transient expression of the L and S subunits in N. benthamiana leaves, allowing for in vitro production of eCPMV nanoparticles. 134 P.H. Beatty, J.D. Lewis / Advanced Drug Delivery Reviews 145 (2019) 130–144 showed a similar biodistribution as the intravenous route except for a exposed CPMV residues are first converted to active ligation handles lower level in brain tissue. CPMV labelled with gadolinium (Gd) chelate by a chemical reaction with N-(4-pentynoyloxy) succinimide to deriva- or Gd+3 and Tb+3 ions complexed directly to the capsid were quantita- tize them with an alkyne group. Then the azide-containing functional tively measured in mice after intravenous administration of one, ten moiety is reacted with the alkyne to perform the click chemistry reac- and 100 mg CPMV per kg body weight [47]. By 20 min after injection tion. Alkyne and azide containing molecules are virtually absent from there were no detectable CPMV particles in the plasma and by 30 min biological systems, so the CuAAC reaction can be considered most of the CPMV particles were found in the liver and spleen. Encourag- bioorthogonal since these two reactive partners will not chemically in- ingly, even after administration of the highest dose of decorated CPMV, teract with other chemicals found within the cell [59]. The allows for the mice showed no toxicity or behavioral changes, although the hema- CuAAC reactions to be done under physiological conditions and in vivo tology analysis showed that the mice were slightly leukopenic [47]. without the production of toxic by-products [59]. Another type of click reaction prevalent in CPMV derivatization is hydrazone ligation be- 2.2. Chemical bioconjugation tween aldehyde groups and either hydrazide or alkoxyamine groups. This derivatization strategy has been successfully used to conjugate a Capsid virus nanoparticles like CPMV can be chemically functional- vascular endothelial growth receptor-1 targeting peptide to CPMV ized on three surfaces of their capsid structure: inside, outside or the nanoparticles, which is discussed further in Section 4.5 [54]. subunit interface [4] (reviewed in [48,49]). Functionalization requires Multi-functionalized CPMV, also termed ‘smart’ tools, allow for the the presence of chemically-reactive ligation handles on the capsid and design of nanoparticles as theranostic tools for use in both diagnosis complementary chemically reactive functional group (s) that will and therapy [20]. These capsids may be conjugated with functional moi- allow for an efficient conjugation reaction to proceed [48,50]. There eties for imaging and targeting and may also carry a drug payload for de- are a few external, solvent exposed, amino acid residues per asymmetric livery to tumor cells (reviewed in [48]). There are multiple different unit that have been experimentally proven to be addressable sites for approaches to making a CPMV nanoparticle smart tool, which really chemical derivatization on the CPMV capsid; 5 lysine residues (300 highlights the versatility of these VNPs. The concentration of activation per capsid), 8 to 9 aspartic and glutamic acid carboxylate groups reagent dictates the number of ligation handles that are activated and (480–540 per capsid) and 2 tyrosine residues (120 per capsid) [11]. available for conjugation, therefore one approach is to activate a fraction The number of internally exposed addressable residues is limited to 2 of the lysine residues at a time by using a low concentration of the acti- cysteine residues per asymmetrical unit, one on the L subunit (Cys vation reagent and then conjugating the chemically reactive functional 295 and one on the S subunit (Cys 4) [11,51]. It is important to remem- groups to these ligation handles in sequential steps [58,68]. Another ber that although viral nanoparticles like CPMV are drawn as inflexible method is “one-pot” chemistry that takes advantage of the orthogonal shells, they are actually dynamic structures that can undergo reversible nature of click chemistry to combine two or more click reactions simul- transitions [51]. The addressable lysine residues (Lys 38, 82, 99, 34 and taneously, such as CuAAc and hydrazone ligation [60]. A totally different 199) have been used extensively in cancer research as ligation handles approach to multi-functionalization was described in Section 2.5.with by derivatizing the side chain amino group with activation reagents the cell-free protein synthesis platform. and so will be the focus of this review (Fig. 1)[33,52,53]. Overall, the activation reagent kinetics, the varying degree of lysine There are two general chemical strategies employed to introduce li- derivatization and the use of orthogonal linkage chemistries have gation handles to the side chains of selected addressable residues; stan- been used advantageously to conjugate the addressable lysine residues dard (also called traditional) chemistry and Click chemistry. These with more than one type of functional group in a graduated, stepwise chemistries derivatize the amino acid residue with a reactive chemical derivatization procedure [54,60]. group that adapts them to become ligation handle sites for functional CPMV conjugation reactions are done in aqueous solutions, there- moiety decoration of the capsid [14,54]. Some commonly used standard fore if the peptide ligand to be displayed on the CPMV external surface chemistry activation reagents include N-hydroxysuccinimidyl ester is hydrophobic in nature its solubility will be low, resulting in low bio- (NHS), maleimide, isothiocyanate and carbodiimide [11,55,56]. The availability, poor conjugation efficiency and unstable CPMV-peptide li- chemical nature of the activation reagent used depends on the chemis- gand display [20]. However, the conjugation efficiency of hydrophobic try of the amino acid residue to be used as the ligation handle (Fig. 3) ligands can be maximized by conjugating the ligands to the terminal [23,55,57]. Standard chemistry activation reagents have slow kinetic ends of PEG polymers so that the hydrophobic ligands are distal to the properties, therefore excessive amounts of the reagent must be used nanoparticle surface. In addition to this tethered distancing, conjugating to ensure that all of the addressable residues are derivatized and can the hydrophobic ligands to PEG first and then the hydrophobic ligand- participate as ligation handles, which limits the functional groups to PEG group to the capsid at the addressable lysine residues using click low molecular weight compounds like dyes [14,58]. The position of chemistry, also maximizes the conjugation efficiency. the lysine in the asymmetric unit also influences derivatization density CPMVs reported in the literature for cancer research have been func- in that some lysine residues are preferentially derivatized before others. tionalized with a variety of different sizes of PEG, ligands, peptides, Click chemistry is a term that describes a set of versatile, covalent or- epitopes, carbohydrates, metal cofactors, synthetic polymers, photosen- ganic reactions that are used to “click” chemical fragments together in a sitizers, and dyes (some of these are depicted in Fig. 3)[2,66]. CPMVs way reminiscent of LEGO pieces to form complex molecules from sim- have also been designed and tested for uses other than cancer diagnosis ple primary chemical components (reviewed in [59,60]). Click reactions and therapy, such as controlled fabrication of CPMV solid support arrays are orthogonal because although the reagents are highly reactive with for receptor-ligand recognition and binding and organometallic moiety each other and form quantitative yields of product, they are also highly CPMV decoration for nanoscale electricity [69,70]. selective and therefore unreactive with a broad range of other func- Fluorescence labelling of CPMVs is verified using UV–vis absorption tional chemical groups under mild conditions [61]. One of the most spectroscopy and the number of dye molecules per capsid (degree of la- often-used types of click reactions for the derivatization of CPMV nano- belling) is calculated using the Beer-Lambert law and the specific ex- particles is the copper (I)-catalyzed azide-alkyne cycloaddition reaction tinction coefficients for the CPMV nanoparticle and the fluorophore (CuAAC) that makes a thermally stable triazole linkage that is inert to [12]. Unlabelled CPMVs show UV–vis absorption peaks at 170 nm and oxidation, reduction and hydrolysis [62–65]. CuAAC has a high thermo- 270 nm. The fluorescently labelled CPMV will be detected by the pres- dynamic driving force which catalyses an irreversible conjugation reac- ence of the absorption peak for its particular fluorescence, for example tion, even with low concentrations of high molecular reagents AF647 dye molecules have one absorption peak at 660 nm. CPMV- [11,66,67]. Therefore, high molecular weight functional groups can be AF647 will therefore show three peaks in the UV–vis spectra; 170 nm, conjugated to addressable capsid residues. The external solvent 270 nm and 660 nm [71]. Functionalized CPMVs can be characterized P.H. Beatty, J.D. Lewis / Advanced Drug Delivery Reviews 145 (2019) 130–144 135

Fig. 3. (a) Traditional NHS activation reagent to derivatize the amine groups of addressable residues for conjugation, (b) ethylene glycol monomer, (c) copper catalyzed Click Chemistry to derivatize addressable residues for conjugation, (d) chemical structure of the modified pan-bombesin peptide, (e) chemical structure of the epidermal growth factor-like domain 7 binding peptide, (f) the C60 (Buckyball) fullerene modified to CPMV and (g) the polyamidoamine dendrimer conjugated to CPMV. 136 P.H. Beatty, J.D. Lewis / Advanced Drug Delivery Reviews 145 (2019) 130–144 via indirect methods like banding patterns with sucrose gradient centri- acids or less and has an isoelectric point of 9 or less [20,26]. These exter- fugation and size exclusion chromatography and they can be visualized nally exposed recombinant peptide sequences have been tested exper- using transmission electron microscopy (TEM) to determine if the cap- imentally as targeting peptides, receptor-association peptides and sid structure is intact after chemical modification [14,43]. Denaturing epitopes for antibody production [26]. SDS-PAGE is also often used to measure the molecular weight of puri- The CPMV VP60 gene sequence has also been genetically modified to fied labelled CPMVs so as to confirm that the functional labels are cova- introduce mutant, surface exposed cysteine residues for use as address- lently attached [14]. For PEGylated CPMV nanoparticles either optical able thiol sites on the capsid [20]. Thiols are reactive groups that can be and chromatographic methods or gravimetric analysis is generally easily functionalized by conjugation with a variety of chemical moieties. used to determine the degree of PEGylation [58,72,73]. These genetically modified CPMV nanoparticles, called CPMVCYS, have been produced that display thiol-reactive fluorescent dyes. However, 2.3. Shielding of CPMV nanoparticles for increased bioretention undecorated, externally exposed thiols tend to form disulfide bridges

with each other, therefore a disadvantage to these mutant CPMVCYS is In vivo studies show that CPMVs elicit an immunogenic response in the ease of which they form interparticle aggregates [20]. Another series humans and that the mammalian reticuloendothelial system clears of genetically modified amino acid residue insertion mutant CPMVs are protein-based nanoparticles from the circulation system before called CPMVHIS. because the modified capsids display 6 sequential histi- reaching the target cells [53,73–75]. To mitigate these undesirable out- dine residues in either the L subunit βE-βF loop or the S subunit βB-βC comes CPMVs are coated with inert polymers that add an immunity- loop or carboxy terminus. The his-tags are surface exposed and address- response shielding ‘stealth layer’ to the particles [7,19]. Polyethylene able and can be derivatized with a variety of functional moieties [11]. glycol (PEG) polymers are commonly used as the stealth layer by conjugating the polymers to external addressable residues on the capsid, a 2.5. Production of CPMV and CPMV virus-like particles process called PEGylation (Fig. 3)[23]. The water soluble, biocompatible, non-ionic PEG polymers comprise different numbers of straight or For in vivo production of CPMV, the leaves of ten-day old branched chain ethylene glycol monomers with various terminus V. unguiculata plants are infected using a mechanical protocol where chemical caps, and therefore differ in size, shape and molecular weight the leaves are lightly dusted with a mixture of the abrasive carborun- [53]. PEGylation has been shown to increase the in vivo half-life of dum and CPMV particles carrying RNA-1 and RNA-2 separately. After CPMV in plasma, extend their circulation time and therefore increase 10 days of plant growth, the CPMV-infected leaves are harvested and accumulation in tumors, a process that is called the enhanced perme- stored at −80 °C [58,80]. ability and retention effect (EPR) [74,76]. These characteristics of The CPMV particles are purified from the aqueous fraction of the ho- CPMVs enable them to be effective tumor imaging and drug delivery mogenized leaves via multiple centrifugation cycles, NaCl-PEG 8000 tools. Further enhancement of these beneficial characteristics has been treatment and ultracentrifugation to form a crudely purified CPMV pel- successfully done by coupling with longer chain or branched PEG poly- let. The CPMV sample is purified to homogeneity via sucrose gradient mers, increasing the density of PEG polymerization on the capsid sur- ultracentrifugation and size-exclusion fast protein liquid chromatogra- face or changing the terminus moiety [73,77]. PEGylation stealth phy (FPLC) [58]. layers can also reduce CPMV off-target actions [8]. The targeting ligands The CPMV RNA-1 and RNA-2 genomes have also been separately on functionalized CPMVs can also be bound to the PEG polymer at either inserted into an Agrobacterium tumefaciens-based plant vector infiltra- the distal or proximal capsid side to increase target recognition or up- tion system used to infect Nicotiana benthamiana leaves [81]. Infection take by the target cell [53,73]. with A. tumefaciens carrying either RNA genome individually did not Recently there have been reports of PEG-specific antibodies found in produce recombinant CPMV particles within the leaves. However, human sera [75]. PEG antibodies would stimulate the hosts' immune when both genomes were agro-infected into the plant leaves at an system to tag the nanoparticles, leading to accelerated blood clearance equal ratio, recombinant CPMV was produced and purified from the (ABC) and reduction of PEGylated nanoparticle efficacy [75]. Alterna- leaves with a yield similar to that from mechanical inoculation of tive, nature-inspired shielding material like carbohydrates (heparin, plant leaves with CPMV. In addition, every inoculated plant became in- hyaluronic acid and other glycosaminoglycans, sialic acid polymers), fected with CPMV showing that the agro-infection method was very ef- lipids (red blood cell membranes, white blood cell membranes, platelet ficient at CPMV production [81]. membranes, synthetic membrane wraps), and proteins (serum albu- Non-replicative CPMV particles, termed virus-like particles min, CD47 cell membrane glycoprotein, elastin-like peptides, synthetic (VLPs) can also be produced by taking advantage of the RNA peptides, zwitterionic peptide) have been conjugated to CPMVs using genome-independent, in vitro self-assembly characteristics of the L click chemistry [75]. These decorated nanoparticles were tested in a va- and S subunits that make up the capsid, via two very different ap- riety of systems for immune recognition avoidance and showed positive proaches; trans expression with high expression plasmids or mixed resultsascamouflage agents [75]. re-assembly of purified subunits in a cell-free protein synthesis platform [28,41,51,82–85]. 2.4. Genetic engineering to introduce functionality The CPMV genes encoding the capsid proteins, as either the full- length RNA-2 sequence or as the partially-processed 60 kDa L and S sub- CPMV separately encapsulates the RNA-1 and RNA-2 genomes that unit fusion protein VP60, and the 24K protease processing enzyme have carry the genetic code for the viral proteins, therefore this technology been cloned into plasmid vectors that allow for high expression of the allows for a genotype-phenotype linkage similar to phage display tech- proteins in either plants or insect cell lines (Fig. 2)[28,78,86]. CPMV nology. DNA encoding the recombinant peptide is cloned into cDNA VLPs can be produced by trans expression of the virally encoded RNA- vectors of the RNA genomes (Fig. 2). Cloning sites for DNA insertion 2 sequence or by VP60 and 24K protease genes in either Spodoptera have been engineered into the VP60 gene so that the translated func- frugiperda insect cells or cowpea plant cell protoplasts. The L and S sub- tional peptide moiety is displayed on externally-exposed β-barrel units are efficiently processed and released by 24K protease cleavage loops, as a chimera [19,26,78]. Functional group exposure via genetic from either the RNA-2 vector or the VP60 vector and they can self- mutational insertion can either display the peptide on the highly assemble in either the plant cell protoplasts or insect cells to form struc- surface-exposed βB-βC and βC’-βC” loops on the S subunit or the less turally intact, RNA-empty VLPs (termed eCPMVs here but an equivalent surface-exposed βE-βF loop on the L subunit [20,79]. The genetically- term used in the literature is CPMV eVLPs, reviewed in [79]) modified, chimeric viral capsid retains form, infectivity and yield com- [27,28,87–89]. The VP60 and 24K protease encoding genes can also be parable to WT-CPMV if the recombinant peptide sequence is 40 amino transiently expressed in Nicotiana benthamiana leaves, mediated by P.H. Beatty, J.D. Lewis / Advanced Drug Delivery Reviews 145 (2019) 130–144 137 the Agrobacterium tumefaciens binary vector transduction system, using nanoparticles lend themselves well for oral delivery because their natu- either the two vectors that together are termed CPMV-HT or using a sin- ral hosts are edible legumes, therefore offering the potential to design gle vector designed for high expression levels called pEAQexpress- edible therapeutics [40]. VP60-24 K that carries the VP60 and 24K protease genes along with Researchers developing CPMV nanoparticles for human cancer ther- P19 gene which reduces gene silencing in the plant (Fig. 2)[28,78,89]. apy use eCPMV nanoparticles as the therapeutic agent. As described in One week after the N. benthamiana leaves have been transformed Section 2.5 and Table 1, eCPMV retain all the well-characterized capsid with the CPMV-HT vectors, the eCPMV particles can be harvested and proteinaceous structural features of CPMV, without the RNA genomes purified. Yields of inplantaproduced eCPMV are very high, at 1 g pure present. This ensures that eCPMV is non-replicative and therefore eCPMV per kg fresh-weight leaf tissue (Table 1)[89]. Genetically mod- non-infectious in vivo [97]. ified eCPMV nanoparticles can also be produced this way by insertion of DNA sequences encoding peptides into the VP60 gene prior to the 3.1. Tumor homing peptides transient N. benthamiana leaf transformation [78,89]. Genetic modification of CPMV RNA is discussed further in Section 2.4. One of the greatest advantages to using nanoparticles in cancer ther- CPMV VLPs can also be made using an in vitro mixed re-assembly apy is the ability to decorate the nanoparticle with a functional group, assay, or cell-free protein synthesis platform, that was originally de- such as a peptide that will guide the nanoparticle directly to the cancer signed with CCMV subunits [85]. In this approach two populations of cells and efficiently differentiate cancer cells from healthy cells in vivo. capsid are functionalized with different display ligands; the capsids There are many peptides described in the literature that have been stud- are disassembled, and the subunits are isolated. Then the subunits ied and tested as targeting molecules and over 700 experimentally val- displaying the different ligands are mixed in a specific ratio and idated tumor homing peptides and covered 23 types of tumors have allowed to re-assemble together to form a mosaic capsid that is been compiled in a database called TumorHoPe that is freely available now displaying both functional ligands [85]. There has been much to researchers that want to design peptide-based drugs and drug- research published recently on the production and use of synthetic, delivery systems [94]. mosaic or hybrid virus-like particles that are a combination of two Although CPMV nanoparticles are plant pathogens, they can natu- or more self-assembling viral capsid proteins and chemically reac- rally home towards mammalian cells; targeting and interacting with a tive polymers [90]. Mosaic capsids have also been made from mixing 54 kD non-glycosylated membrane protein initially called CPMV bind- CPMV subunits and synthetic polymers like the dendrimer ing protein (CPMV-BP) and recently identified as surface exposed- polyamidoamine (PAMAM) that is described in Section 3.4.These membrane bound vimentin [98,99]. Native tropism towards vimentin hybrid VLPs can exhibit enhanced scaffold and cargo vesicles is discussed in more detail in Section 4.3. properties. Many types of human cancers, such as those found in ovaries, brain, Comparison of the crystal structure of purified eCPMV to the crystal kidney, breast, myeloid cells and lung, express and display high levels of structure of RNA-containing WT-CPMV and to the cryo-electron micros- folate receptors on their surfaces [100,101]. For some cancers, such as copy structure of eCPMV shows that they are identical in form [41]. ovarian, folate receptor overexpression is linked with a higher histologic eCPMV nanoparticles offer more benefits to their use than just their grade [100]. Folic acid-PEG decorated CPMV nanoparticles were able to non-replicative nature; they may have improved capsid surface modifi- differentially target tumor cells versus healthy cells in vivo. Research is cation chemistries and they can take up a larger range of cargo molecule ongoing into developing folate-conjugated CPMV nanoparticles that can types [89]. Some examples of the design and use of CPMV VLPs in cancer target and internalize high grade ovarian cancer tumors in order to de- research are discussed in this review. liver anticancer drugs [100].

3. Therapeutic and theranostic uses of CPMVs 3.2. Cargo loading

There are different types of cancer treatments available currently, CPMVs can stably carry molecules within its protein capsid, differen- dependent on the location and type of cancer with the most common tiate between healthy and cancerous host cells, be taken up and accu- therapies involving highly toxic or invasive methods of chemotherapy, mulate inside of tumor cells and release their cargo molecules once surgery and radiotherapy. Improvements have been made to these tra- they have been internalized [27]. These features make CPMVs very ef- ditional treatments that include laser surgery and fluorescent imaging fective at delivering toxic molecules into tumor cells while avoiding guided surgery, and new anti-cancer drugs have been developed, such the healthy host cells and so these particles are promising therapeutic as doxorubicin and STAT3 inhibitors [88,91]. VNPs, such as RCNMV, vectors in cancer treatment. have also been developed to deliver doxorubicin to cancer cells for ther- The CPMV capsid naturally has funnel shaped channels, that are 7.5 apeutic procedures [92,93]. As well, novel tumor homing peptides are A in diameter at the external, narrow end, at the 5-fold axes points on being discovered, experimentally validated and developed that can tar- the scaffold that appear to be permeable to various molecules [26,87]. get different cancer cell types for imaging and therapy [94,95]. Cur- CPMV naturally encapsulates one of the two viral RNA genomes. These rently, nanotechnology cancer therapies are being designed and nucleic acids have been used to help load cargo molecules with a revers- studied extensively because they show great promise as improved ible affinity for nucleic acid, for example dyes such as DAPI and drug delivery vectors with high specificity and high sensitivity in prefer- proflavine that intercalate with nucleic acid or positively charged mole- entially targeting cancer cells, even within deep, hard-to-reach tissues cules with an electrostatic attraction to RNA. The non-covalent cargo [95]. Additionally, multifunctional nanoparticles, CPMV included, are load infusion capacity rate of RNA-containing CPMV particles was deter- being designed as theranostic tools that combine imaging and therapy mined to be 10,000 fluorophores per nanoparticle for 1 h. Empty CPMV moieties in one device to provide effective detection of, and drug deliv- nanoparticles (i.e. no RNA genome) were not able to load the tested ery to, cancer cells in vivo [88]. fluorophores into the capsid. The infused CPMVs were dialyzed and cen- The bioavailability of CPMVs after administration via intravenous in- trifuged through spin filter columns to remove excess, externally- jection or orally in mice models have also been tested and the nanopar- associated cargo molecules, and the load capacity was measured at ticles were found in nearly all tissues after both delivery methods 130 to 155 dye or drug molecules per CPMV nanoparticle, dependant [23,43,96]. In particular, with oral delivery, CPMV nanoparticles were on the cargo molecule size [27]. This equated to a 50–70% recovery stable under gastric conditions and because they were detected yield of released cargo relative to the amount of starting material used. throughout the mice, CPMV appeared to have crossed the gastrointesti- RNA genome-empty CPMV (eCPMV) can be infusion-loaded with nal epithelium via interactions with Peyer's patches [43]. CPMV cargo that does not require nucleic acid interaction for payload trapping 138 P.H. Beatty, J.D. Lewis / Advanced Drug Delivery Reviews 145 (2019) 130–144

[87]. In particular, eCPMV nanoparticles have been used to load cobalt or PS characteristics, improved water solubility and biocompatibility as iron oxides in a benign process that does not alter capsid size or mono- well as evidence of accumulation within HeLa cells in an in vitro assay dispersion properties and the external, addressable amino acid residues when imaged by confocal microscopy [103]. can still be conjugated with functional moieties [87]. The CPMV interior Zinc ethynylphenyl porphyrin (ZnEpPor) is a novel PS in the same has a net negative charge due to glutamic and aspartic acid residues, chemical family as the often-used clinical PS, Photofrin. In vitro macro- which entraps the metal ions via electrostatic interactions [87]. This phage staining assays and murine melanoma studies showed that technology has been propelled forward by improvements to generating ZnEpPor is a more potent PS than Photofrin because of its enhanced ac- large amounts of eCPMV nanoparticles in plants without viral infection, cumulation in tumor tissues and better binding efficiency, due to its which would require the bipartite genome [28,87]. The electrostatic in- positive charge and zinc (II) cation, respectively [84]. When ZnEpPor teraction between the capsid interior and metal ions also make the is used as a PS in PDT treatment, macrophage and tumor cells are elim- cargo stably loaded, even after 6 h of dialysis against buffers [87]. The inated, however enhancing this PS would greatly improve the ZnEpPor- internally-loaded cobalt or iron oxide CPMV nanoparticles are detect- based PDT treatment. CPMV addressable lysine residues were able by transmission electron microscopy (TEM) without staining, derivatized with an azide linker, then conjugated via click chemistry while the non-loaded eCPMV are not [87]. The metal ion cargo is also to alkyne derivatized carbonyl dendrons [84]. Carbonyl dendrons are chemically reactive still with cobalt participating in reduction reactions synthetic poly-branched polymers with carbonyl termini that allow enabling them to act as possible metal particle-nucleation sites and iron for conjugation with carbohydrates. They were designed for use in diag- oxide able to undergo autocatalytic hydrolysis [87,102]. nostic tools, regenerative medicine and nanobiotechnology applica- Cargo loading via covalent conjugation of the payload to the internal tions, in order to exploit the highly specific recognition processes side of CPMVs has also been attempted [88]. CPMVs covalently loaded between glycans and their receptors [84]. Glycoconjugates can be easily with the therapeutic drug doxorubicin resulted in successful tumor pen- produced on dendrons via carbonyl chemistry. The CPMV-dendron con- etration, tumor cell internalization and targeting of the loaded CPMVs to jugate (CPMV*) has many more addressable sites for ligand display than the cellular endolysosome, but cargo release only occurred as the inter- CPMV alone. CPMV* and ZnEpPor PS were mixed to allow electrostatic nalized CPMV particles were slowly metabolized, over a few days, by the interactions to form PS-CPMV*. PS decorated CPMV* showed improve- tumor cells [88]. In contrast, the non-covalently loaded fluorophores ments in both macrophage and cancer cell elimination compared to were released from the internalized nanoparticles within 60 min [27]. ZnEpPor [84]. Besides the obvious advantage of a fast release from non-covalently bound, infusion-loaded CPMV payload, the other advantages to this 3.4. Use of CPMV as a vaccine in cancer immunotherapy method include; expansion in the chemical diversity of the payload from amino acid containing cargo to virtually any type of molecule, nei- One reason for tumor proliferation throughout the body is because ther the cargo or the capsid require chemical or structural modification the tumor cells mediate a tumor-localized suppression of the immune upon loading and no system is needed for efficient cargo release [27]. system. This de-sensitizes the host immune system to tumor specific antigens which allows the tumors to grow and metastasize unchecked.

3.3. Photodynamic therapy using CPMV-C60 fullerene conjugates Immunotherapies designed to reverse tumor-mediated immune suppression have been studied to determine their potential efficacy as anti- Photosensitizer chemicals will produce reactive oxygen species tumor treatments [71,83,97]. (ROS) after they are irradiated with a specific wavelength of visible Both wildtype and RNA genome-free CPMVs, especially non- light that matches the absorption spectrum of the specificphotosensi- PEGylated capsid particles, will elicit an immune response when tizer [103]. ROS are strong oxidizing agents that quickly oxidize introduced into mammalian tissues. These nanoparticles show a natural (remove electrons) DNA, proteins, lipids and carbohydrates, ultimately tropism towards immune cells; in specific, the M2 subpopulation of causing cell death. Photodynamic therapy (PDT) is a minimally-invasive macrophages, although the mechanism of the attraction is not under- therapy used to treat localized, shallow tumors like highly resistant and stood yet [97]. These features, along with the ability of CPMV and aggressive melanoma, leukemia T cells, prostate cancer cells and CD22 eCPMV to penetrate tumors and specifically target cancer cells, make + human lung cancer cells [84]. In PDT, a photosensitizer (PS) is admin- them good candidates for use as in situ immunostimulatory nanoparti- istered to the tumor tissue, then the patient is irradiated with visible cle vaccines aimed at re-sensitizing the host immune system to tumor light to catalyze the ROS production. PDT has been shown to kill cells specific antigens without needing to know what antigens are present via the ROS formation, but it also stimulates an immune response by [71]. eCPMV anti-tumor vaccines are intravenously injected into an al- the host, both of which help to kill the cancer cells. These excellent out- ready identified tumor where they stimulate the hosts' innate and comes of PDT are unfortunately tempered by limitations of the PS. These then adaptive immune infiltration and activation; in effect priming the chemicals tend to be inefficient at tumor-targeting, leading to off-target hosts' immune system against the tumor and turning it from a “cold” effects, have low accumulation within the tumors, low solubility under to a “hot” tumor [97]. physiological conditions leading to formation of colloidal aggregates Non-modified eCPMV nanoparticles were able to trigger the and low to moderate light absorption properties [84,91]. mouse innate and adaptive immune system, in particular the acti-

The Buckminsterfullerene carbon allotrope C60 (buckyball), is an ex- vated neutrophil population was stimulated, after in situ injection cellent free radical scavenger and therefore highly useful as a PS, but it is into the intraperitoneal (IP) space next to disseminated tumors at also hydrophobic and bio-incompatible in physiological conditions multiple anatomical sites [83]. The eCPMV vaccination was admin- [103]. CPMV nanoparticles are highly biocompatible, can effectively tar- istered to a variety of mouse metastatic cancer tumors in vivo, in- get and accumulate in tumors and tumor cells. Researchers theorized cludingB16F10lungmetastatic-like melanoma, dermal B16F10 that conjugating these two structures could result in an enhanced PS melanoma, 4T1 BALB/c syngenic breast cancer model that metasta- with improved bio-compatibility and targeting characteristics. C60 sizes to the lung, intradermal CT26 colon tumor and disseminated buckyballs were successfully conjugated to CPMVs using click chemistry peritoneal serous ovarian carcinoma [83]. In all of these tumor

(CPMV-C60) and cellular uptake of these functionalized capsids were models the tumors were either signiﬁcantly reduced in size or measured using the HeLa human cancer cell line [103]. When the de- even eliminated altogether (dermal B16F10 melanoma) after in gree of derivatization was analysed the C60 moiety was only found situ eCPMV administrations. The eCPMV-mediated anti-tumor ef- linked to lysine residue 38 (K38) on the large subunit, which was previ- fect was experimentally determined to be due to immune stimula- ously determined to be a highly reactive, solvent exposed, lysine tion and not from tumor cell cytotoxicity because in vitro

(Fig. 1). Even with the low degree of C60 decoration, CPMV-C60 showed application of the eCPMV particles to the tumor cell lines showed P.H. Beatty, J.D. Lewis / Advanced Drug Delivery Reviews 145 (2019) 130–144 139 no effect on cancer cell proliferation or viability [83]. However, the CPMVs can be decorated with hundreds of gadolinium ions (Gd) by in situ eCPMV vaccination needed to be repeated weekly to replen- derivatizing the addressable lysine residues, therefore enabling CPMV- ish the nanoparticles to a therapeutic concentration to continue re- Gd particles to present with high relaxivity values [55,64]. High sensitiv- sensitizing the mouse immune system towards the ovarian tumors ity and high-resolution imaging of tumor neovasculature and blood [83]. Although eCPMV nanoparticles are very effective in situ flood using CPMV nanoparticles functionalized with fluorescent dyes immune-stimulatory agents, especially with the metastatic ovarian and with, or without targeting ligands, have been done using a spinning cancer model, the administration of weekly, repeated IP vaccina- disk confocal fluorescence microscope, capturing three-dimensional Z tions into human tumors, especially in deep, hard-to-reach loca- stacks as well as time-lapse images [20,23,76,106]. tions like ovarian cancer tumors, is problematic because they The multivalent display capacity of CPMV nanoparticles provides the require hospitalization. sensitivity and specificity needed to detect these cancers, while their In order to improve the in vivo retention time of the eCPMV vaccine nanoscale size allows these particles to extravasate through the leaky after tumor-site in situ administration, and reduce the number of puta- tumor vasculature, penetrate into deep tissue locations and ultimately tive in-hospital injections, a mosaic eCPMV particle was developed [71]. be taken up by, and accumulate in, the targeted cancer cells, via the The mosaic nanoparticles were generated by mixing the L and S sub- EPR effect [76,107]. units from CPMV and PAMAM dendrimer polymers together and The biological models used to determine the efficacy of experimen- allowing them to re-assemble into a novel nanoparticle called eCPMV- tal CPMVs in vivo include the chick embryo chorioallantoic membrane G4. These mosaic eCPMV-G4 nanoparticles showed increased retention (CAM), mouse embryo and adult mice. time in vivo which allowed the nanoparticle concentration to remain at therapeutic levels for much longer than eCPMV without G4 [71]. 4.1. Chick embryonic chorioallantoic membrane models PAMAM dendrimers are positively charged, nanoscale polymers that consist of an ethylenediamine core with radiating, branched amido- Imaging cancer cell proliferation, invasion and mobility in live ani- amine arms that terminate with primary amine groups [71]. They are mal models is a powerful method to study tumor growth and experi- used in medicine and industry as synthetic nanoparticles [5]. One ad- mental diagnostic and therapeutic targets. Intravital imaging has been ministration of eCPMV-G4 to the IP cavity in a mouse disseminated limited however by the lack of efficient probes and animal models. Re- ovarian cancer model showed the same level of efficacy as an anti- cently, both WT and fluorescent dye and target peptide-decorated tumor vaccine as the weekly administration of the soluble CPMV [71]. CPMVs have shown to be excellent imaging probes [23,25,54,80]. The This confirmed that the combination of the PAMAM dendrimer and chorioallantoic membrane (CAM) of chick embryos is a very useful tis- eCPMV produces a novel mosaic nanoparticle that acts as a reservoir sue model for nanoscale, in vivo, non-invasive, real-time, intravital of eCPMV in the IP cavity. eCPMV is therefore slowly released to provide imaging of tumor growth. Human tumors from cell lines; HEp3 (squa- a continuous supply of immunostimulatory nanoparticles into the ovar- mous carcinoma), HT29 (colon adenocarcinoma), HT1080 (fibrosar- ian cancer tumor, which triggers the host immune response to recog- coma), MDA-MB-231 (breast carcinoma), and PC3 (prostate cancer), nize the tumor specific antigens immunogenic [71]. Reducing the have been successfully grafted onto the CAM of shell-less chick embryos number of vaccine administrations while maintaining a high concentra- [58]. High-resolution images of these tumors were obtained via confocal tion of the immunotherapeutic agent at the tumor site is essential for microscopy because the human tumors proliferate laterally and shal- beneficial patient care and quality of life. lowly on the membrane, to an average depth of 200 nm. The CAM Researchers have also compared the efficacy of in situ injected to- model is relatively inexpensive to use, the xenograft procedure is bacco mosaic virus nanoparticles (TMV) to stimulate the hosts' immune done without the need for anesthetic or surgery and a fully vascularized response against melanoma tumors versus the efficacy of eCPMV [104]. human tumor can be developed on the CAM within 7 days [76]. The eCPMV nanoparticles outperformed short-rod, long-rod, spherical, CPMV particles of varying sizes and ligand chemistries have been assembled-protein and free protein TMV variants, as measured by re- injected into the CAM at sites distal to the tumor xenograft where duction of tumor volume after administration. This differential potency they flow through the bloodstream and can extravasate from the may be because eCPMV is more effective at recruiting monocytes into leaky tumor vasculature and also be quickly taken up by endothelial the tumor microenvironment which also attracts tumor infiltrated neu- cells. This allows for the tumor vasculature to be labelled from both trophils and natural killer cells plus the production of effector memory within and outside of the tumor. Additionally, CVMP nanoparticles can cells [104]. be stably maintained within tumor grafted CAM for up to 72 h for imag- The development of thermostable and chemical-resistant viral bio- ing and quantification measurements. logical agents would greatly benefit their use as vaccines. CPMV nanoparticles have been successfully biomineralized into calcite crystals 4.2. Deep tissue imaging using CPMVs decorated with multivalent fluores- that can protect the spherical capsid from harsh chemical environments cent dyes [105]. It is essential to use non-invasive techniques to visualize tumor en- 4. Intravital vascular imaging for non-invasive cancer detection dothelial cells and tumor remodelling of vascular tissue in vivo, to better understand where and when tumor neovascularization occurs. In vivo Ultrasound (US) and computed tomography (CT) are the conven- penetration of deep tissues with fluorescent dyes for imaging tumors tional imaging technologies used for detection, diagnosis, triaging and is very challenging [23]. CPMVs conjugated with fluorescent dye mole- prognosis of organ cancers and tumor metastasis [76]. Although these cules can show a high signal to capsid ratio which allows for imaging of technologies have been proven to be very effective cancer research rare or deep cellular targets. CPMV nanoparticles, displaying up to 120 and clinical tools, they lack the resolution required to image small pri- fluorescent dye molecules per capsid (i.e. CPMV-A555), injected mary lesions at the microscopic level [20,76]. Magnetic resonance imag- into adult mice were found to localize to the mouse vascular endothe- ing (MRI), positron emission tomography (PET) and spinning disk lium and internalize, via endocytosis, within endothelial cells as confocal fluorescence microscopy are non-invasive, in vivo imaging monodispersed particles (Fig. 4)[23]. This preferential targeting and in- tools that are used together with a contrasting agent, such as gadolin- ternalization of endothelial cells allows for easy identification of the ium ion tracers, to generate bright images with high sensitivity [50]. tumor-associated venous vessels, which in turn helps determine the CPMV nanoparticles have been successfully developed as contrasting vascular origin and directionality [23]. However, the exact mechanisms agent platforms that increase the density and allow for targeting of that allow for specific cell targeting and internalization between CPMVs the specific cells. Due to their large surface area to volume ratio, and the targeted antigen-presenting cells (APCs) are not fully 140 P.H. Beatty, J.D. Lewis / Advanced Drug Delivery Reviews 145 (2019) 130–144

Fig. 4. Intravital imaging using engineered CPMV nanoparticles. (a) Visualization of individual head and neck HEp3 cancer cells (green) invading along a blood vessel (red) labelled with CPMV-A555 in the avian embryo CAM. (b) Intravital imaging of a mature blood vessel in the avian embryo CAM with CPMV-A555 (red) and CPMV-A488 (green) injected at distinct time points. Endothelial cell nuclei are visualized using Hoechst 33342. (c) Dual fluorescent CPMV labeling of blood vessel with CPMV-A555 (red) and surrounding lymph vessels with CPMV- A488 (green) in the avian embryo CAM. (d) Intravital imaging of human breast cancer tumors (MDA-MB468, green) in the chorioallantoic membrane using PEGylated CPMV VNPs functionalized with RGD peptides (red) over 12 h. understood [108]. Research aimed at elucidating these processes will microvasculature by CPMV-A555 during in vivo intravital imaging of greatly benefit the development of CPMV nanoparticles as vaccines both mouse and chick embryo models was superior to other dye- and tumor cell-targeting tools. CPMV used as vaccines is discussed fur- displaying nanospheres (Fig. 4)[23]. ther in Section 3.4 and CPMC tropism for vimentin-displaying cancer cells is outlined in Section 4.3. 4.3. Native tropism of CPMV for vimentin on host cells As mentioned in the previously, CPMVs are stable within tissues for up to 72 h, therefore long-term, real-time intravital imaging with fluo- Vimentin is a type III intermediate filament mainly expressed in the rescently labelled CPMV nanoparticles can allow for quantification cytoplasm of mammalian mesenchymal cells. In wildtype cells, cyto- of the endothelial cells and mapping of the neovasculature (Fig. 4) plasmic vimentin is a cytoskeleton protein filament that takes part in [23,57]. This differential cellular targeting and long-term stability wound healing, cell-cell interactions, motility, contraction, proliferation could allow researchers to determine which tumor vascular system is and molecular functions like transcription, translation, signal transduc- the source of tumor cells metastasizing into the hosts circulatory system tion and apoptosis [20,55]. Cytoplasmic vimentin is also a part of the cell [23]. PEGylated CPMVs that were also conjugated to the fluorophore adhesion-matrix adhesion complex which makes it an important pro- FITC were blocked from internalization within the endothelial cells, sug- tein for migrating cells, suggesting that cytoplasmic vimentin plays gesting that the capsid structure that interacts with the cancer cell re- a role in tumor metastasis [109,110]. Vimentin is a mesenchymal ceptor, possibly vimentin, is obstructed when PEG polymers decorate cell marker and a potentially important component in epithelial– the capsid externally [23]. The resolution of macro and mesenchymal transition (EMT) events, which has been linked to P.H. Beatty, J.D. Lewis / Advanced Drug Delivery Reviews 145 (2019) 130–144 141 tumor invasiveness and aggressiveness [111]. Wildtype and cancerous so that new blood vessel production is always on. This has the effect mammalian cells also express surface-displayed vimentin for an, as of nourishing the tumor cells with the elements and molecules required yet, unknown function but inflamed endothelial cells associated with for cell growth and increasing the likelihood of metastatic tumor cell tumors can be differentiated from wildtype cells because they over- dispersal to other parts of the body. Inhibition of tumor angiogenesis express surface-displayed vimentin [111]. In clinical studies, vimentin (tumor neovascularization) would stop the tumor cell nutrient supply overexpression by breast cancer cells is linked to invasiveness and and the dispersal of tumor cells throughout the body, therefore this pro- poor prognosis for patients [20]. The experimental and clinical evidence cess is considered an important anti-tumor therapeutic target [54,113]. on vimentin and cancer cells points to surface-displayed vimentin as a A report was recently published that outlined the use of a human- good target for tumor cell localization and imaging. ized anti-VEGF monoclonal antibody (called Bevacizumab) in an anti- Wildtype CPMV displays natural tropism towards vimentin- angiogenic immune-therapy that has been approved for treatment of expressing cells in vitro [1]. Specifically, CPMV shows a high affinity glioblastoma, non-small cell lung cancer, metastatic colorectal cancer for surface displayed vimentin but this affinity is significantly reduced and metastatic renal cell cancer, as either a stand-alone drug or in com- after labeling CPMV with PEG2000 [20,108]. Despite this low affinity, bination with other drugs [116]. In an effort to selectively target VEGFR- CPMV labelled with either the fluorescent dye A647 (CPMV-A647) or 1, a high affinity VEGFR-1 interacting peptide was found (called F56) both A647 and PEG2000 (P2-A647) were able to target and be taken using phage-display and was conjugated to CPMV to produce VEGFR-1 up by vimentin-expressing human colon adenocarcinoma cells (HT- targeting nanoparticles [54]. The CPMV-F56 nanoparticles (called FP3) 29) in vivo after a longer incubation time. In fact, P2-A647 uptake by was generated using hydrazine ligation chemistry and is an excellent the tumor cells was three times higher than CPMV-A647. Other recent example of a ‘smart’ multifunctional nano-platform because not only studies have shown that low affinity ligands appear to target tumor does it display 133 copies of the VEGFR-1 selective peptide, it also was cells better than their high affinity counterparts and this may be due decorated with over 140 fluorescent dye molecules for imaging and to the EPR effect [112]. It was theorized that P2-A647 showed high up- over 50 PEG polymers to lengthen the plasma circulation time [54]. take by vimentin-expressing cancer cells despite its low affinity for two The in vivo targeting and imaging capability of FP3 was measured in reasons; because it extravasated in the tumor-associated blood vessels HT-29 tumor-bearing mice, known to express high levels of VEGFR-1. and more importantly, the circulation half-life was increased due to Two hours after administration of FP3, the tumors were immune- the PEGylation stealth layer [20]. Therefore, fluorescent dye-labelled fluorescently stained and imaged using confocal microscopy and FP3 CPMVs have been effectively used to find and visualize tumor- was found accumulated throughout the tumor tissue but not on the en- associated endothelial cells and tumor neovasculature by targeting to dothelial cells that line the blood vessels [54]. Therefore, VEGFR-1 ap- surface-displayed vimentin [12,20]. pears to be expressed on both endothelial cell and tumor cell surfaces but due to FP3 extravasating via the EPR effect from the leaky tumor 4.4. CPMV targeted to gastrin-releasing peptide receptors blood vessels to the tumor cells, decorated CPMVs were not detected on the endothelial cells [54]. Gastrin-releasing peptide (GRP) receptors are over-expressed on many different tumor cell types and so its efficacy as a highly specific 4.6. Neovascular imaging via epidermal growth factor-like domain 7 target for tumor detection and imaging was tested using CPMVs as the (EGFL7) protein targeting delivery tool. CPMV was multivalently functionalized using the externally addressable lysine residues and a combination of standard conju- Despite the VEGF anti-angiogenic therapies under development or gation chemistry and click chemistry to display Alexa Fluor (AF) 647, available for therapeutic use, clinical evidence is mounting to suggest PEG and a 14 amino acid peptide analogue of pan-bombesin [20]. The that targeting VEGF may not be effective in inhibiting tumor neovascu- PC-3 prostate cancer tumor was grafted onto the CAM and the CPMV- larization [25]. The 30 kDa epidermal growth factor-like domain 7 pro- AF647-PEG-bombesin nanoparticle was introduced at the distal side of tein (EGFL7) is only expressed by endothelial cells undergoing vascular the tumor. The CPMVs accumulated in 5 mm wide tumors in the CAM remodelling and not by quiescent cells and has been identified as a test. The observation that nanoparticles can home onto rare target key regulator of several angiogenic pathways [25]. Evidence has cells using the EPR effect gives credence to the theory that the EPR effect accumulated to implicate EGFL7 as a key factor in tumor angiogenesis occurs with small tumors and metastatic lesions [20]. and therefore this protein may be a good candidate as an anti- angiogenesis target. In mice studies, over-expression of EGFL7 led to ab- 4.5. Neovascular imaging via vascular endothelial growth factor receptor normal vasculature remodelling; similar to tumor-related abnormal (VEGFR) targeting blood vessel remodelling. Solid tumor in vivo studies detected high amounts of EGFL7 protein, which have also been positively co- Angiogenesis, the de novo formation of new blood vessels, is a com- correlated with high tumor grades and poor prognosis in breast cancer, plex, multi-pathway process that in normal, adult physiology occurs hepatocellular carcinoma, laryngeal squamous cell carcinoma, malig- when the body repairs tissue after wounding or during placental forma- nant glioma, ovarian cancer and pancreatic cancer [25]. tion with pregnancy [25,113]. The formation of new blood vessels is CPMVs displaying EGFL7-targeting peptide ligands, called E7p72, highly regulated at the transcriptional level in wildtype cells, in part were developed to determine the validity of EGFL7 as a target for the de- by a multitude of different growth factors and their respective receptors, tection and imaging of tumor-related, active vasculature remodelling such as the well-studied vascular endothelial growth factor (VEGF) and [25]. Similar to the FP3 nanoparticle described above, the E7p72 deco- VEGF receptors 1, 2 and 3 (VEGFR-1, -2 and 3) [54,114,115]. VEGFR-2 rated CPMV nanoparticle was developed as a multifunctional platform and -3 are over-expressed in tumor vasculature and so have been pop- [25]. The resulting CPMV displayed Alexa-Fluor 647 near infrared dye ular targets, both individually and in combination, for non-invasive molecules and PEG-E7p72 polymers conjugated with E7p72 displayed imaging and targeting of solid tumor endothelial cells in the neovasc- on the distal side of the capsid (CPMV-PEG-E7p72). These nanoparticles ulature [113,115]. VEGFR-1 has been identified as a tumor-specific vas- were tested for targeting and imaging efficacy using an in vitro endothe- cular endothelial cell surface protein by subtractive proteomic mapping lial cell plate assay with confocal microscopy and an in vivo mouse and is expressed in tumor cells in breast cancers, gastric cancers and model and chick embryo-CAM study and, both xenografted with the schwannomas [54]. Therefore VEGFR-1 may also be a useful target for HT1080 fibrosarcoma cell line. The xenografted tumors were assayed tumor imaging and targeting. Tumor angiogenesis is characterized by to positively determine that the tumor neovasculature associated endo- abnormal vasculature and a poor cancer prognosis with high mortality. thelium cells expressed high levels of EGFL7 [25]. ex vivo staining During cancerous tumor growth, angiogenic pathways are deregulated of mouse tumor cryo-sections with CPMV-PEG-E7p72 showed that 142 P.H. Beatty, J.D. Lewis / Advanced Drug Delivery Reviews 145 (2019) 130–144 the decorated nanoparticles were bound to the tumor blood vessel cells. Competing interests statement Time-lapse intravital imaging of intravenously injected CPMV-PEG- E7p72 CAM xenografted tumors revealed that the decorated The authors declare no competing financial or non-financial nanoparticles accumulated in endosomal compartments of the tumor interests. endothelium cells over a 90-minute time duration but not within non- tumor endothelial cells [25]. 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